Journal of Experimental Botany, Vol. 68, No. 10 pp.

24772488, 2017
doi:10.1093/jxb/erx101 Advance Access publication 17 April 2017

REVIEW PAPER

Nitrogen use efficiency in crops: lessons from Arabidopsis

andrice
HuaLi1,2, BinHu1 and ChengcaiChu1,*
1
State Key Laboratory of Plant Genomics and CAS-JIC Centre of Excellence for Plant and Microbial Science (CEPAMS), Institute of
Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
2
University of Chinese Academy of Sciences, Beijing 100039, China

* Correspondence: ccchu@genetics.ac.cn

Received 14 November 2016; Editorial decision 2 March 2017; Accepted 4 March 2017

Editor: Alain Gojon, INRA

Abstract
Application of chemical fertilizers, especially nitrogen (N), to crops has increased dramatically in the last half cen-
tury and therefore developing crop varieties with improved N use efficiency (NUE) is urgent for sustainable agri-
culture. N utilization procedures generally can be divided into uptake, transport, and assimilation. Transporters
for nitrate or ammonium acquisition and enzymes for assimilation are among the essential components determin-
ing NUE, and many transcription factors also play a pivotal role in regulating N use-associated genes, thereby
contributing to NUE. Although some efforts in improving NUE have been made in various plants, the regulatory
mechanisms underlying NUE are still elusive, and NUE improvement in crop breeding is very limited. In this review,
the crucial components involved in N utilization and the candidates with the potential for NUE improvement in
dicot Arabidopsis and monocot rice are summarized. In addition, strategies based on new techniques which can
be used for dissecting regulatory mechanisms of NUE and also the possible ways in which NUE can be improved
in crops are discussed.

Key words: Ammonium, Arabidopsis, genetic improvement, GWAS, nitrate, nitrogen, nitrogen use efficiency, rice, transporters.

Introduction
A marked increase in food production has occurred over the
past half-century to feed the growing population, which is
mainly attributed to the application of chemical fertilizers
(Godfray etal., 2010; Liu etal., 2013). However, excess input
of fertilizers also results in severe environmental pollution,
climate change, and biodiversity loss, which is an enormous
environmental challenge of the 21st century. Nitrogen (N) is a
major driving force for crop yield improvement. It is estimated
that >120 Mt of N fertilizer is used annually and less than half
the N applied in developing countries was taken up by crops
(Rothstein, 2007; Kant etal., 2011). Moreover, the application
of N fertilizer will keep increasing with the growing demand
for food production. Thus, reducing fertilizer usage but main-
taining crop yield is a pressing challenge. One of the ideal ways
to tackle this crisis is to improve N use efficiency (NUE) of
crops. It is estimated that ~US$1.1 billion could be saved annu-
ally with a 1% increase in NUE (Kant etal., 2011).
The definition of NUE is complex (Xu et al., 2012), but
it can be simply defined as the yield of grain, forage, or fruit
per unit of N available in the soil according to different plant
species. To improve the NUE in crops, it is important to gain
a better understanding of the function and regulation mecha-
nism of the key components involved in N acquisition, trans-
port, assimilation, and signal transduction.

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 2478 | Li et al.

N uptake and transport in Arabidopsis
andrice
Plants are immobile organisms which have evolved several N
uptake systems to support their survival in the changing envi-
ronment. Nitrate and ammonium are the two main inorganic
N forms which exist in soils, of which nitrate is the major form
of N available in aerobic environments, whereas ammonium
tends to be the major form in flooded environments or acidic
soils. Amino acids also account for a small portion of organic N
that can be also utilized by plants (Nsholm etal., 2009; Moran-
Zuloaga etal., 2015). In agriculture practice, urea is the major
N fertilizer and it could be rapidly degraded into ammonium
and carbon dioxide by urease in the soils, although a small por-
tion of urea can be absorbed directly by plants (Liu etal., 2003;
Kojima etal., 2006; Arkoun etal., 2012; W.H. Wang etal., 2012).
Nitrate transporters in Arabidopsis
In Arabidopsis, there are four families of nitrate transporters,
NPF (NRT1/PTR), NRT2, the chloride channel family (CLC),
and slow anion channel-associated homologues (SLAC/
SLAH), with 53 NPF, 7 NRT2, 7 CLC, and 5 SLAC/SLAH
members, respectively; ~20 members have been identified and
characterized so far. Among the NRT1 family genes charac-
terized, some can transport nitrate or dipeptides, while some
can also transport auxin, abscisic acid (ABA), jasmonoyl-iso-
leucine, gibberellin (GA), or glucosinolates (Krouk etal., 2010;
Kanno etal., 2012; Nour-Eldin etal., 2012; Y.Y. Wang etal.,
2012; Weichert etal., 2012; Boursiac etal., 2013; Lran etal.,
2014, 2015; Pellizzaro etal., 2014; Saito etal., 2015; Tal etal.,
2016. Little is known so far with regard to why some nitrate
transporters can transport different substrates even when the
substrates have large difference in their structures. Figure 1
summarizes all N transporters characterized based on their
physiological functions in both Arabidopsis andrice.
AtNPF6.3 (CHL1/AtNRT1.1) was the first nitrate trans-
porter identified to function not only in nitrate uptake (Tsay
et al., 1993) but also in nitrate translocation from roots to
shoots (Lran et al., 2013). It was also found to be a dual-
affinity nitrate transporter, and the switch between the high-
and low-affinity form is determined by the phosphorylation
of Thr101 (Liu and Tsay, 2003). Recently, the crystal structure
of AtNRT1.1 has been resolved by two independent groups
(Parker and Newstead, 2014; Sun et al., 2014; Tsay, 2014);
Parker and Newstead drew a model whereby phosphorylation
of AtNRT1.1 can increase structural flexibility and result in
different transport rates, while Sun and colleagues support the
idea that there is a phosphorylation-controlled dimerization
switch which allows AtNRT1.1 to have distinct affinity modes.
In addition, AtNRT1.1 can repress lateral root development at
low nitrate concentrations and it is proposed that AtNRT1.1
can transport basipetal auxin out of lateral roots to repress
root growth (Krouk etal., 2010; Bouguyon etal., 2015).
In addition to AtNRT1.1, AtNPF4.6 (AtNRT1.2) and
AtNPF2.7 (NAXT1) are also involved in root nitrate uptake,
with influx and efflux activity, respectively (Huang et al.,
1999; Segonzac et al., 2007; Kanno et al., 2012). AtNPF7.2

Fig.1. Integrative model to illustrate physiological functions of nitrate and ammonium transporters in Arabidopsis and rice. (a) Detailed illustration of
nitrate and ammonium uptake or translocation in Arabidopsis. NPF6.3 (CHL1, NRT1.1), NPF4.6 (NRT1.2), NRT2.1, NRT2.2, NRT2.4, and NRT2.5 are
involved in nitrate uptake from the external environment. NPF2.7 (NAXT1) mediates nitrate efflux. NPF2.3, NPF7.3 (NRT1.5), NPF7.2 (NRT1.8), and
NPF2.9 (NRT1.9) play a role in regulating root to shoot nitrate transport. NRT2.4, NRT2.5, NPF1.1 (NRT1.11), NPF1.2 (NRT1.12), and NPF2.13 (NRT1.7)
play a role in source to sink nitrate remobilization. NPF2.12 (NRT1.6) functions to deliver nitrate from maternal tissue to the developing embryo. NRT2.7
transports nitrate into the seed vacuole, and NPF5.5 affects N accumulation in the embryo. AMT1.1, AMT1.2, AMT1.3, and AMT1.5 participate in
ammonium uptake, AMT1.4 is expressed in pollen. (b) Detailed illustration of nitrate and ammonium uptake or translocation in rice. NRT2.1, NRT2.2,
NRT2.4, NPF6.5 (NRT1.1B), NPF8.9 (NRT1), and NPF2.4 are involved in nitrate uptake. NPF2.2, NRT2.3a, NPF6.5, and NPF2.4 participate in root to
shoot nitrate translocation. NPF4.1 (SP1) is expressed in the panicle and controls panicle size. AMT1, AMT2, and AMT3 are involved in ammonium
uptake, while the data only come from the expression status. Different coloured tagged characters or arrows indicate different transporters and their roles
in corresponding physiological processes. HATS, high-affinity transport system; LATS, low-affinity transport system.

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(AtNRT1.8), AtNPF7.3 (AtNRT1.5), AtNPF2.3, and
AtNPF2.9 (AtNRT1.9) are major transporters for long-dis-
tance nitrate transport. AtNRT1.8 is expressed predominantly
in xylem parenchyma cells within the vasculature and is impor-
tant for xylem nitrate unloading (Li et al., 2010), whereas
AtNRT1.5 is responsible for xylem nitrate loading, and these
two transporters mainly mediate long-distance root to shoot
transport (Lin etal., 2008). Asimilar expression pattern in roots
was observed for AtNPF2.3 which contributes to nitrate secre-
tion into root xylem sap under salt stress (Taochy etal., 2015).
AtNRT1.9 mediates nitrate loading into root phloem to influ-
ence nitrate translocation and distribution (Wang and Tsay,
2011). AtNPF6.2 (AtNRT1.4) is found to play a special role in
nitrate homeostasis between the petiole and leaf lamina (Chiu
etal., 2004). AtNPF2.13 (AtNRT1.7) is expressed preferentially
in the phloem of leaf minor veins and is responsible for nitrate
remobilization from source to sink (Fan etal., 2009). AtNPF1.1
(AtNRT1.12) and AtNPF1.2 (AtNRT1.11) are also found to
facilitate nitrate redistribution from mature leaves to young
tissues (Hsu and Tsay, 2013). AtNPF2.12 (AtNRT1.6) is only
expressed in the reproductive tissues, and its mutation leads to
accumulation of less nitrate in the mature seeds (Almagro etal.,
2008). Another seed expressed transporter, AtNPF5.5, affects
N accumulation in the embryos (Lran etal., 2015).
In contrast to the transporters in the NRT1 family, the mem-
bers of the NRT2 family transport nitrate with high affinity.
The NAR2-like family genes are auxiliary partners for high-
affinity nitrate uptake (Kawachi etal., 2006; Okamoto etal.,
2006; Orsel etal., 2006; Laugier etal., 2012; Yong etal., 2010).
Disruption of AtNRT2.1 and AtNRT2.2 impairs nitrate
uptake in both the inducible high-affinity transport system
(IHATS) and the constitutive high-affinity transport system
(CHATS) (Li etal., 2007). AtNRT2.4 is induced by low nitrate
and mediates nitrate transport under N starvation in both
shoots and roots (Kiba etal., 2012). AtNRT2.5 plays an essen-
tial role in N-starved adult plants by ensuring efficient uptake
of nitrate and by participating in nitrate loading into the
phloem during nitrate remobilization (Lezhneva etal., 2014;
Kotur and Glass, 2015). Moreover, AtNRT2.5 and AtNRT2.6
are involved in Rhizobacterium-stimulated lateral root growth
(Kechid etal., 2013). In particular, AtNRT2.7 is localized to
the tonoplast and is important for nitrate storage in seed vacu-
oles (Chopin et al., 2007). Similarly to AtNRT2.7, AtCLCa
and AtCLCb are also found to mediate nitrate accumula-
tion in vacuoles (Geelen etal., 2000; De Angeli etal., 2006;
von der Fecht-Bartenbach et al., 2010). AtCLCc was found
by quantitative trait locus (QTL) analysis of nitrate storage
in a recombinant inbred line (RIL) population to be located
in a major QTL (Harada etal., 2004). AtSLAC1, AtSLAH2,
and AtSLAH3 show the ability to be nitrate-permeable anion
channels (Geiger et al., 2011; Maierhofer et al., 2014), and
AtSLAH3 participates in alleviation of ammonium toxicity in
a nitrate-dependent manner (Zheng etal., 2015).
Nitrate transporters inrice
In rice, there exist >80 NRT1/PTR, 4 NRT2, and 2 NAR2
members, but only a few members of the NRT1/PTR family
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Improvement of nitrogen use efficiency in crops | 2479
have been characterized to date (Araki and Hasegawa, 2006;
Cai et al., 2008; Feng et al., 2011). OsNPF8.9 (OsNRT1)
is the first low-affinity nitrate transporter identified in rice
which mediates nitrate uptake in roots (Lin et al., 2000).
Overexpression of OsNRT1 can accumulate more N in rice
plants only under high N conditions, but expression of its
splicing form with six transmembrane domains can accumu-
late more N in plants and promote rice growth under both
low and high N conditions (Fan et al., 2015). OsNPF4.1
(SP1), is a putative PTR gene, which is highly expressed in
the phloem of young panicle branches to control panicle size,
but neither nitrate nor any other substrate transport activity
was detected (S. Li etal., 2009). Overexpression of OsNPF7.3
(OsPTR6) significantly improves rice growth but has little
function in NUE improvement (Ouyang et al., 2010; Fan
etal., 2014). OsNPF8.20 (OsPTR9) is regulated by different
N sources and its overexpression improves production, but
fails in transporting di-/tripeptides, amino acids, nitrate, or
ammonium (Fang etal., 2013). Knockout of OsNPF2.4 leads
to decreased low-affinity nitrate acquisition in roots, impaired
K-coupled nitrate root to shoot transportation, and reduced
nitrate redistribution from old leaves to N-starved roots or
young leaves (Xia et al., 2015). osnpf2.2 mutants are defec-
tive in long-distance nitrate transport, with repressed nitrate
unloading from the xylem, resulting in plant growth retarda-
tion and impact on grain filling (Li etal., 2015).
OsNPF6.5 (OsNRT1.1B) is predominantly expressed in
root hairs, epidermis, and stellar cells adjacent to the xylem
in roots, and the osnrt1.1b mutant showed defects in both
nitrate uptake and root to shoot nitrate transport, suggesting
that OsNRT1.1B is involved in nitrate uptake and transport.
Interestingly, phylogenetic analysis showed that OsNRT1.1B
diverged between indica and japonica subspecies, and the
OsNRT1.1B indica genotype has undergone positive selec-
tion during domestication. NUE in the japonica near isogenic
line (NIL) carrying the indica-type allele improves dramati-
cally and subsequently leads to an actual yield increase of
30.333.4% under low N conditions (Hu etal., 2015).
Regarding high-affinity nitrate transporters in rice, four
NRT2 and two NAR2 genes have been identified so far.
OsNRT2.1, OsNRT2.2, and OsNRT2.3a need to interact
with OsNAR2.1 for nitrate uptake, while OsNRT2.3b and
OsNRT2.4 can function independently without NAR2 (Araki
and Hasegawa, 2006; Cai etal., 2008; Feng etal., 2011; Yan
etal., 2011). OsNRT2.3a is mainly expressed in xylem paren-
chyma cells of root steles, and knockdown of OsNRT2.3a
impairs xylem loading of nitrate as well as decreased plant
growth at low nitrate supply, demonstrating a role in long-dis-
tance nitrate transport from root to shoot (Tang etal., 2012),
while the other spliced form, OsNRT2.3b, is mainly expressed
in the phloem and possibly functions as a pH sensor (Fan
etal., 2016).
Ammonium transporters in Arabidopsis andrice
In addition to nitrate, ammonium is another important
N source for plant growth, especially when plants such
as rice grow under anaerobic conditions. Phylogenetic
 2480 | Li et al.
analysis shows that the Arabidopsis ammonium transporters
AtAMT1;1AtAMT1;5 have the highest homology to each
other and cluster with cyanobacterial ammonium transport-
ers, while only AtAMT2 is similar to Saccharomyces cerevi-
siae Mep1, 2, and 3 and Escherichia coli AmtB (von Wirn
etal., 2000; Loqu and von Wiren, 2004; Ho and Tsay, 2010).
AtAMT1;1, AtAMT1;2, and AtAMT1;3 are all highly
expressed in the roots. AtAMT1;1 and AtAMT1;2 can also
be found in shoots, with the highest expression in mature
leaves. Enhanced ammonium uptake is correlated with up-
regulated AtAMT1;1 and AtAMT1;3 under N deficiency
(Gazzarrini et al., 1999). The atamt1;1 mutant displayed
~3040% reduced high-affinity root ammonium transport
under N-limited conditions, and a highly lethal phenotype
when grown with ammonium and sucrose (Kaiser et al.,
2002). The atamt1;1 atamt1;3 double mutant shows an
additive effect of these two ammonium transporters (Loqu
etal., 2006). It was found that AtAMT1;1 and AtAMT1;3
could form functional homo- and heterotrimers in plant
roots to regulate ammonium transport activity (Yuan etal.,
2013). It has been further shown that AtAMT1;3 is clus-
tered and internalized through clathrin-mediated endocytic
pathways under high ammonium levels (Wang etal., 2013),
which provides an effective way for plants to avoid excess
ammonium accumulation. AtAMT1;2 is localized in the
endodermis and root cortex, and high-afnity inux was
reduced by 1826% in the atamt1;2 mutant compared with
the wild-type plant, suggesting that AtAMT1;2 mediates
ammonium uptake when it enters the root via the apoplas-
mic transport route (Yuan etal., 2007). An amt1;1 amt1;2
amt1;3 amt2;1 quadruple mutant (qko) showed severe
growth retardation under ammonium supply and main-
tained only 510% of total high-afnity ammonium uptake
capacity. In qko, the transcription level of AtAMT1;5 was
up-regulated in N-decient rhizodermal and root hairs, sug-
gesting that AtAMT1;5 accounts for the remaining ammo-
nium uptake capacity (Yuan et al., 2007). In contrast to
all the other AMT transporters, AtAMT1;4 is exclusively
detected in the microsomal membrane fractions of flower
tissue and probably makes a contribution to N nutrition
during pollen development (Yuan etal., 2009).
Although ammonium has been recognized as the major
form of N nutrition in rice, only limited information has so
far been documented on ammonium transporters, mainly
in terms of the expression pattern, localization, or trans-
port activity (Suenaga et al., 2003; Sonoda et al., 2004;
Ferreira etal., 2015; Yang etal., 2015). Rice has 12 puta-
tive ammonium transporters which can be divided into
five groups (von Wirn et al., 2000; Suenaga et al., 2003;
B.Li etal., 2009; Li etal., 2012). OsAMT1;1 is expressed
in both roots and shoots, and is induced by ammonium.
The mutants of OsAMT1;1 showed decreased ammonium
uptake as well as repressed growth of roots and shoots
under both low and high ammonium conditions (Li etal.,
2016). OsAMT1;2 is induced by ammonium exclusively in
roots, while OsAMT1;3 is induced by N deficiency in roots.
It was shown that OsAMT1 genes do not depend on the
endogenous ammonium level but rather on the glutamine
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concentration, which indicates that glutamine, the prod-
uct of ammonium assimilation, reflects the intracellular N
status (Sonoda et al., 2004). OsAMT2.1 is constitutively
expressed in both roots and shoots irrespective of the N
source. OsAMT3.1 is relatively weakly expressed (Suenaga
etal., 2003). Considerable efforts have been made to improve
rice ammonium transport efficiency by overexpressing dif-
ferent OsAMT genes, with very little success (Hoque etal.,
2006; Ranathunge etal., 2014; Bao etal., 2015).
N assimilation in Arabidopsis andrice
After nitrate is absorbed into roots, it is first reduced to nitrite
by cytosolic nitrate reductase (NIA). Then nitrite is imported
into the plastids and further reduced to ammonium by nitrite
reductase (NIR). Two NIA genes exist in Arabidopsis (Cheng
etal., 1991; Wilkinson and Crawford, 1991, 1993). AtNIA2 is
a major isoform in the leaves and its mutant retains only 10%
of wild-type NIA activity (Wilkinson and Crawford, 1991).
Furthermore, nia1 nia2 double mutants have only 0.5% of
wild-type NIA activity and display poor growth (Wilkinson
and Crawford, 1993). Three NIA genes exist in rice (Hamat,
1989), and NIA-deficient rice mutants showed only ~10%
leaf NADH-NIA activity of that of the wild type (Hasegawa
etal., 1991, 1992).
Glutamine synthetase (GS), a key enzyme in N assimila-
tion and remobilization, forms the GSGOGAT cycle with
glutamate synthase (GOGAT) to convert inorganic ammo-
nium into glutamine. There are two isoforms of GS, cytosolic
GS1 and plastidic GS2. Cytosolic GS1 is responsible for pri-
mary ammonium assimilation in the roots or re-assimilation
of ammonium produced in the leaves during protein turno-
ver, while GS2 is primarily responsible for assimilation of
ammonium produced from photorespiration in chloroplasts
(Thomsen etal., 2014). Though GS is important in N assimila-
tion, it has a relatively small gene family with 35 members in
various plants (Bernard and Habash, 2009; Swarbreck etal.,
2011; Goodall etal., 2013; Betti etal., 2014). In Arabidopsis,
GLN1;1, GLN1;2, and GLN1;4 are induced during leaf
senescence (Guo etal., 2004); GLN1;2 is the only one which is
significantly up-regulated by ammonium and localized in the
vasculature, GLN1;3 is also located in the vasculature, while
its expression is restricted to the root mature zone. GLN1;1 is
located at the root surface, root tips, and root hairs. GLN1;4
is specifically expressed within the basal region of lateral root
emergence. The GLN1;5 transcript has not been detected so
far (Ishiyama et al., 2004). gln1;2 mutants displayed lower
GS activity, higher ammonium concentration, and reduced
rosette biomass under ample nitrate supply, as well as longer
root hairs under sole ammonium conditions (Lothier etal.,
2011).
In rice, there are three GS members, OsGS1;1 and OsGS1;2
are expressed in all organs and showed a reciprocal response
to ammonium supply in the cell layers of the root surface,
whereas OsGS1;3 is specifically expressed in the spikelet and
functions in assimilation of ammonium into maturing seeds
(Tabuchi etal., 2007). Mutation of OsGS1;1 resulted in severe
retardation in growth rate and grain filling (Tabuchi etal.,

2005), whereas osgs1;2 mutants displayed severe reduction
in active tiller number (Funayama etal., 2013). In contrast
to osgs1;1, a remarkable increase in free ammonium ions
was observed in osgs1;2 roots, which indicates an important
role in the primary assimilation of ammonium uptake by
roots. osgs1;3 apparently exhibited a reduced rate of natural
senescence in the paddy field (Yamaya and Kusano, 2014).
It has been shown that overexpression of the soybean or
pea GS1 gene in tobacco produces growth advantages by
improvement of the photosynthetic/photorespiratory pro-
cess (Fuentes etal., 2001; Oliveira etal., 2002). It was also
demonstrated that overexpression of Gln1-3 in maize leaves
can increase kernel number by 30% (Martin et al., 2006).
Rice overexpressing OsGS1;1 or OsGS1;2 showed elevated
GS activities as well as soluble protein concentrations, but
failed to show an increase in grain yield (Cai etal., 2009),
while another study showed that overexpression of OsGS1;2
could improve NUE under growth chamber conditions, but
has no effect under greenhouse conditions (Brauer et al.,
2011).
There are also two kinds of GOGAT, ferredoxin-dependent
(Fd-GOGAT) and NADH-dependent (NADH-GOGAT)
enzymes, which differ in electron donor specificity (Lancien
etal., 2002). In Arabidopsis, Fd-GOGAT accounts for the
majority of leaf GOGAT activity and its mutant shows
severe chlorosis and growth defects (Somerville and Ogren,
1980). GLU1 and GLU2 are two Fd-GOGATs identified in
Arabidopsis. GLU1 is expressed abundantly in the leaves
while GLU2 is mainly expressed in the roots (Coschigano
et al., 1998). Mutant analyses reveal that GLU1 plays a
major role in photorespiration and N assimilation in leaves,
whereas GLU2 may have a major role in primary N assimi-
lation in roots (Coschigano et al., 1998). GLT is the only
NADH-GOGAT gene in Arabidopsis, and it functions in
non-photorespiratory ammonium assimilation and glu-
tamate synthesis (Lancien et al., 2002). Two NADH-type
GOGATs and one Fd-GOGAT exist in rice (Tabuchi etal.,
2007). Similar to OsGS1;1, OsNADH-GOGAT2 is local-
ized in vascular tissues of mature leaves and its mutation
caused marked reduction of spikelet number and hence
productivity (Tamura et al., 2011), suggesting their co-
ordination in N remobilization from leaves to seeds during
leaf senescence. On the other hand, OsNADH-GOGAT1,
like OsGS1;2, is also expressed in the surface cell layers of
roots in an ammonium-dependent manner, and its mutants
displayed a similar alteration in the content of amino acids
and free ammonium ions to the osgs1;2 mutants (Tamura
et al., 2010). Overexpression of the NADH-GOGAT gene
with its own promoter from japonica rice in an indica cul-
tivar produced an increase in grain weight (Yamaya etal.,
2002).
Besides GS/GOGAT, some enzymes responsible for N
assimilation such as glutamate dehydrogenase (GDH), aspar-
tate aminotransferase (AspAT), and asparagine synthetase
(AS) were also reported to play important roles in N metab-
olism and improving NUE in plants (Brears et al., 1993;
Ameziane etal., 2000; Carvalho etal., 2003; Lam etal., 2003;
Wong etal., 2004).
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Improvement of nitrogen use efficiency in crops | 2481
N signalling and transcriptional regulation
N perception
Besides facilitating nitrate uptake, AtNRT1.1 plays dual roles
in nitrate transport/sensing (transceptor) in the perception of
external nitrate concentrations (Ho etal., 2009). Under low
nitrate conditions, a threonine residue (T101) of AtNRT1.1
is phosphorylated by CIPK23 (a CBL-interacting protein
kinase) and triggers a low level primary response. T101 will
be dephosphorylated and leads to a high level primary nitrate
response under high nitrate conditions (Ho et al., 2009;
Bouguyon etal., 2015). AtNRT2.1 might be another nitrate
sensor in Arabidopsis as its mutant has been shown to be
defective in nitrate-triggered root development (Little etal.,
2005; Remans et al., 2006b). Similarly, AtAMT1.1 can be
phosphorylated on a threonine residue in an ammonium con-
centration- and time-dependent manner, leading to co-oper-
ative allosteric effect to avoid ammonium toxicity (Lanquar
et al., 2009; Loqu et al., 2007). These results indicate that
ammonium sensing might employ ammonium transporters
with a similar mechanism to that suggested for nitrate sens-
ing. OsNRT1.1B is one of the rice homologues of AtNRT1.1
and also plays a role in nitrate uptake and translocation in
rice. Interestingly, the induction of OsNIA1 and OsNIA2 by
nitrate is attenuated in the osnrt1.1b mutant, indicating that
OsNRT1.1B also functions as a nitrate sensor or transceptor,
similar to AtNRT1.1 (Hu etal., 2015).
Transcription factors (TFs) in NUE regulation
In addition to the transporters and enzymes mentioned above,
a number of regulatory TF genes are found to participate in
NUE regulation (Table1). It has been shown that transgenic
Arabidopsis or rice expressing maize Dof1 (ZmDof1) could
up-regulate genes associated with organic acid metabolism,
thus increasing N assimilation and enhancing growth under
low N conditions (Yanagisawa et al., 2004; Kurai et al.,
2011). Overexpression of rice Dof TF OsRDD1 can also
increase the uptake and accumulation of various nutrients,
as well as N responsiveness and grain productivity (Iwamoto
and Tagiri, 2016). The GATA TFs GNC (GATA, Nitrate-
inducible, Carbon-metabolism-involved) and CGA1/GNL
(Cytokinin-responsive GATA 1/GNC-like) also play impor-
tant roles in modulating N assimilation, chloroplast develop-
ment, and starch production, which greatly contributes to the
metabolism balance between N and C (Hudson etal., 2011).
Recently, Arabidopsis ELONGATED HYPOCOTYL5
(HY5), a light-responsive bZIP TF, was found to be a shoot
to root mobile signal that mediates C assimilation and trans-
location in the shoots, and activates AtNRT2.1 expression as
well as N uptake (X.B. Chen etal., 2016).
There are also some TFs mainly involved in regulating genes
in N transport or assimilation. In Arabidopsis, RWP-RK
domain-containing proteins AtNLP6 and AtNLP7 have
been shown to be positive regulators of nitrate-induced gene
expression (Castaings et al., 2009; Konishi and Yanagisawa,
2013). Overexpression of AtNLP7 leads to enhanced N
assimilation by co-operatively modulating a number of nitrate
 2482 | Li et al.

Table1. Genes involved in NUE improvement in Arabidopsis and rice

Genes Host plants Strategies used Effects References

OsNPF8.20 (OsPTR9) Rice Ubi1 promoter Promoted lateral root formation, Fang etal. (2013)
increased NH4+ uptake and grain
yield
OsNPF7.3 (OsPTR6) Rice Ubi promoter Increased growth at different N Fan etal. (2014)
supplies, decreased NUE at high
NH4+
OsNPF8.9 (OsNRT1) Rice Ubi promoter Increased biomass under Fan etal. (2015)
hydroponic conditions
OsNPF6.5 (NRT1.1B) Rice NIL, CaMV 35S or native promoter Increased grain yield and NUE Hu etal. (2015)
OsNRT2.1 Rice Ubi or NAR2.1 promoter pUbi:OsNRT2.1 decreased NUE, J.G. Chen etal.
pOsNAR2.1:NRT2.1 increased (2016)
NUE
OsNRT2.3a Rice CaMV 35S/Ubi promoter No increase in yield or NUE Fan etal. (2016)
OsNRT2.3b Rice CaMV 35S/Ubi promoter Improved growth, yield, and NUE Fan etal. (2016)
OsAMT1.1 Rice Ubi1 promoter Increased NH4+ uptake and Hoque etal. (2006);
grain yield under suboptimal and Ranathunge etal.
optimal N conditions (2014)
OsAMT1.3 Rice CaMV 35S promoter Poor growth, C and N metabolic Bao etal. (2015)
status distorted
OsGS1;1 Rice CaMV 35S promoter driven Decreased grain yield Cai etal. (2009)
OsGS1;2 Rice CaMV 35S/Ubi promoter Decreased grain yield/ Cai etal. (2009);
improved NHI and UtE, no NUE Brauer etal. (2011)
improvement under limited N
OsGS2 Rice CaMV 35S promoter Enhanced photorespiration and Hoshida etal. (2000)
salt tolerance
OsNADH-GOGAT Rice Rice NADH-GOGAT promoter Enhanced grain filling Yamaya etal. (2002)
ASN1 Arabidopsis CaMV 35S promoter Enhanced seed protein content, N Lam etal. (2003)
limitation tolerance of seedlings
ASN2 Arabidopsis CaMV 35S promoter Increased Asn content under Igarashi etal. (2009)
normal nutrient conditions
AspAT Arabidopsis/rice CaMV 35S promoter Increased leaf AAT activity, seed Murooka etal. (2002);
amino acid and protein contents Zhou etal. (2009)
OsAlaAT Rice Root-specific promoter OsANT1 Increased tiller number, grain yield Shrawat etal. (2008);
and total plant N Beatty etal. (2009)
E.coli glnA Rice CaMV 35S promoter Decreased grain yield Cai etal. (2009)
ZmDof1 Arabidopsis/rice Maize Ubi promoter/35SC4PPDK promoter Increased N assimilation and Yanagisawa etal.
enhanced growth under low N (2004); Kurai etal.
condition (2011)
ANR1 Arabidopsis Root specific GAL4-GFP transactivator/ Increased length and number of Gan etal. (2012)
Dex-inducible ANR1-GR LRs
AGL21 Arabidopsis CaMV 35S promoter Higher LR density, longer LR Yu etal. (2014)
length
AtTGA4 Arabidopsis CaMV 35S promoter Improved nitrate transport and Zhong etal. (2015)
assimilation activity
OsRDD1 Rice Maize Ubi1 promoter Increased uptake and Iwamoto and Tagiri
accumulation of various nutrients, (2016)
N responsiveness, and grain
productivity
AtNLP7 Arabidopsis CaMV 35S promoter Improved plant growth by Yu etal. (2016)
enhancing N and C assimilation

AAT/AspAT, aspartate aminotransferase; AS, asparagine synthetase; Dex, dexamethasone; GR, glucocorticoid-binding domain; LR, lateral
root; NHI, N harvest index; OsAlaAT, rice alanine aminotransferase; UtE, spikelet yield/shoot N content.

transport, metabolism, and signalling-related genes, and thus
increased biomass (Yu et al., 2016). In contrast to AtNLP7,
AtLBD37/38/39 (Lateral organ Boundary Domain 37/38/39)
are negative regulators in nitrate uptake and assimilation,
and also suppress a typical N starvation response such as
anthocyanin biosynthesis when N is sufficient (Rubin et al.,
2009). The central clock gene CCA1 has a specific regulatory
function in N assimilation genes and can be induced by organic
N metabolites. Overexpression of this gene would repress the
expression of ASN1 and GDH1 and induce the expression

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of GLN1.3 (Gutierez et al., 2008). TGA1 and TGA4 were
identified using an integrative bioinformatics approach as
putative regulatory factors that mediate nitrate responses in
Arabidopsis roots. They can specifically regulate the expres-
sion of nitrate transporters AtNRT2.1 and AtNRT2.2, and
their mutants exhibit nitrate-dependent primary and lateral
root growth impairment (Alvarez et al., 2014).
AtANR1, a MADS box gene, was shown to be involved
in lateral root elongation (Zhang and Forde, 1998), and
might function downstream of AtNRT1.1 as the expres-
sion of ANR1 was dramatically altered in chl1 mutants
(Remans et al., 2006a). Mutation in another MADS box
gene, AtAGL21, leads to impaired lateral root development
especially under N-deficient conditions through regulation
of auxin signalling (Yu etal., 2014). Besides this, there exist
more TFs involved in phytohormone signalling pathways.
The auxin AFB3 regulatory network is found to be involved
in lateral root growth in response to nitrate (Vidal etal., 2010,
2013). By using enhancing regions of NIA1 and NRT2.1 pro-
moters, AtTCP20 was identified and AtTCP20 could also
bind to type-A ARR5/7, thus influencing N signalling and
cytokinin signalling (Guan et al., 2014; Ruffel et al., 2014,
2016). Though quite a number of TFs have been identified
as regulating N utilization or signalling in Arabidopsis, very
little work has been done in rice or other crops.
Strategies for NUE improvement and future
perspective
Although substantial progress has been made in under-
standing the mechanism underlying N utilization, NUE
improvement promoted by this knowledge is still limited. The
transporters have been thought to be the key components
determining NUE, but AtNRT1.1-overexpressing transgenic
Arabidopsis plants showed increased nitrate uptake without
alterations in biomass or seed yield (Liu et al., 1999). In con-
trast, several nitrate transporters have been successfully used
in NUE improvement in rice. For example, overexpression of
OsNPF8.20 results in increased lateral root formation and
ammonium uptake, and finally leads to increased tiller num-
ber and effective panicle number, and thus improved NUE
and grain yield (Fang et al., 2013). It was found that both
OsAMT1;2 expression and GS activity were improved during
the grain-filling stage in OsNPF8.20-overexpressing plants
(Fang et al., 2013). Transgenic plants harbouring OsNRT2.1
under control of its constitutive promoter led to a decrease
in grain yield; however, the transgenic plants harbouring
OsNRT2.1 under control of the OsNAR2.1 promoter could
up-regulate its expression specifically in the roots and culms
and greatly increase NUE and grain yield (J.G. Chen et al.,
2016). The results suggest that specific expression of a target
gene in a certain tissue has to be taken into account in the
manipulation of nitrate transporters. Furthermore, trans-
genic lines harbouring OsNRT1.1B under the control of
both the Cauliflower mosaic virus (CaMV) 35S promoter and
its native promoter showed improved grain yield and NUE
(Hu et al., 2015). It is noteworthy that OsNRT1.1B has an
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Improvement of nitrogen use efficiency in crops | 2483
important role in regulating nitrate signalling and could acti-
vate not only nitrate absorption, but also nitrate assimilation,
which both contribute to NUE improvement.
Nitrate and ammonium are two main forms of inorganic N
for plants, and nitrate is the major N source for land plants,
while ammonium is important for plants such as rice which
grow under anaerobic conditions. It was found that overex-
pression of OsNRT2.3b not only improved nitrate transport
capacity, but also increased ammonium uptake, which is lost
in OsNRT2.3a or the pH sensor domain-mutated overexpres-
sors. Interestingly, plants overexpressing the short form of
OsNRT2.3b increased total N, P, and Fe uptake by influenc-
ing the pH homeostasis and thus benefiting C metabolism,
and finally resulted in improved grain yield as well as NUE
by 40% (Fan et al., 2016). These results indicated that the
pH-buffering ability of OsNRT2.3b is very important for
rice adapting to the ammoniumnitrate shift environment
between flooded and drained soils, and this mechanism may
not exist in land plants such as Arabidopsis. Although over-
expression of transporter genes provide a promising strategy
in improving NUE in crops, it has to be noted that increased
N application would delay flowering, which significantly
increases the risk of yield losses, especially in high-latitude
regions where late-season temperatures can severely affect
grain filling. Thereby, the influence of N on delaying flower-
ing times needs to be considered when overexpression of N
transporter genes is used as a strategy for improving NUE
ofcrops.
The different results obtained in Arabidopsis and rice
with manipulation of transporters may be due to the differ-
ent environments in which they live. Arabidopsis lives in an
aerobic environment with nitrate as the main N source, and
nitrate transport systems might have been highly evolved or
optimized to adapt to its N requirement, whereas rice lives
in paddy fields in which ammonium is the major N source,
and it consequently has evolved a high-efficiency ammo-
nium transport system. Thus, little effect will be obtained
when ammonium transporters are used for manipulation of
NUE in rice. Possibly, it would also be unsuitable to employ
nitrate transporters for NUE improvement because of the
highly evolved nitrate transport system in Arabidopsis. On
the other hand, there exist many N-associated transporters
in plants, with different tissue or cell and even subcellular
localization, and expression patterns greatly determine their
functions. Therefore, precise spatialtemporal expression of
a transporter is important in genetic manipulation of plants.
It should also be noted that a nitrate transporter, in some
cases, may not only mediate the transport process, but also
function as a regulator to control downstream N assimila-
tion or metabolism. Therefore, a full understanding of all
expression patterns and functions for each transporter in
both Arabidopsis and rice will help us to manipulate NUE
in crops precisely.
Due to the difficulties in phenotyping, it has long been
a big challenge to fine-map NUE-related QTLs. By using
chlorate, the toxic analogue of nitrate, to perform phenotyp-
ing, Hu etal. (2015) identified OsNRT1.1B, a critical QTL
contributing to NUE divergence between rice subspecies,
 2484 | Li et al.
by map-based cloning, which provides a potential strategy
for NUE phenotyping in crop plants. It is thus necessary to
work together with chemists to identify new toxic analogues
of different N sources for efficient phenotyping screening in
NUE-related genes/loci. On the other hand, with develop-
ment of new measuring techniques, the high-throughput and
precise phenotyping of NUE-related traits is now possible,
which may also facilitate the cloning of NUE genes/QTLs.
Combined with available re-sequencing data of crop culti-
vars, landraces, and wild accessions, the complex traits could
be identified by genome-wide association studies (GWAS),
which have been successfully applied in Arabidopsis, rice,
maize, and other plant species (Atwell etal., 2010; Huang
etal., 2010, 2011; Korte and Farlow, 2013; Li etal., 2013;
Ogura and Busch, 2015; Si et al., 2016). For example,
JASMONATE RESPONSIVE 1 (JR1) was found to be the
functional gene which was characterized by GWAS using
complex root traits among 96 Arabidopsis accessions in two
N environments (Gifford etal., 2013). With a similar tech-
nique, CALCIUM SENSOR RECEPTOR, PhzC, ROOT
SYSTEM ARCHITECTURE 1, and PHOSPHATE 1 were
discovered by high-throughput automated root image anal-
ysis (Gifford et al., 2013; Rosas et al., 2013; Slovak et al.,
2014). Even at the cellular level, KURZ UND KLEIN, an
F-box family gene, was identified as a novel regulator of
root development using semi-automated confocal micros-
copy (Meijn et al., 2014). These new measuring methods
can be exploited to capture natural variations systematically
under different N regimes, which is extremely helpful for
identifying the N use-associated genes and NUE improve-
ment incrops.
It is worth mentioning that integration of transcriptomics,
proteomics, and metabolomics, which reflect gene expression
or plant molecular phenotypes, may allow us to identify sys-
temically candidate genes or pathways involved in agronomic
traits. In addition, computational biology and systems biol-
ogy also facilitate finding critical genes during domestication.
Using genomic data of different rice accessions, the ammo-
nium transporter OsAMT1;1 shows a much lower genetic
diversity than O. rufipogon which extends to an ~100 kb
genomic region around OsAMT1;1, indicating that this locus
was subjected to strong selection during rice domestication
(Ding etal., 2011). The genes such as OsNRT2.3, OsNAR2.2,
OsGS1;2, or OsGS1;3 have also been shown to have been
selected during domestication (Xie etal., 2015). These results
indicated that selective sweep analysis may help us to better
understand the genes with the potential for NUE improvement
in crops. An idea which is more challenging but worth trying
is to develop crops with the ability to fix N biologically, which
might be achieved by expression of a nitrogenase enzyme in
the crops or by transferring symbiotic bacteria with N-fixing
ability (Beatty and Good, 2011; Rogers and Oldroyd, 2014).
On the other hand, members of the root microbiota pro-
vide additional functions to host plants through facilitating
nutrient acquisition from soil for plant growth. Significant N
fixation under field conditions has been observed in rice, sug-
gesting the great promise of microbiota in improving NUE
(Bulgarelli et al., 2013). Although the composition of rice
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on 06 September 2017
root microbiota is already clear, functions and co-operation
among these microbes are largely elusive (Edwards, 2015).
Now, systematic bacterial isolation approaches are available
combining metagenomics to analyse functionally the plant
microbiota in depth (Bai etal., 2015), which will help us to
reveal the contribution of the root microbes to nutrient solu-
bilization, absorption, or N fixation for plants.
Manipulation of NUE in crops appears to be the best way
to guarantee global food security. With the advent of genome
editing technology, systematic investigation of the genes
detected by GWAS, selective sweeps, or other computational
methods is becoming possible, which will definitely facilitate
transferring our knowledge from theory to practice in crop
NUE improvement.
Acknowledgements
This work was supported by grants from the National Natural Science
Foundation of China (grant no. 31500975), the National Key Research and
Development Program for Seven Crop Breeding Pilot Project (grant no.
2016YFD0100706), and the 973 Program from the National Basic Research
Program of China (grant no. 2015CB755700).
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